Gas filled liposomes and stabilized gas bubbles and their use as ultrasonic contrast agents

ABSTRACT

Contrast agents for ultrasonic imaging comprising gas filled liposomes prepared using vacuum drying gas instillation methods, and gas filled liposomes substantially devoid of liquid in the interior thereof, are described. Methods of and apparatus for preparing such liposomes and methods for employing such liposomes in ultrasonic imaging applications are also disclosed. Also described are diagnostic kits for ultrasonic imaging which include the subject contrast agents.

RELATED APPLICATION

This application is a Divisional of Ser. No. 08/088,268 filed on Jul. 7,1993 and now U.S. Pat. No. 5,348,016 which in turn is a Divisional ofSer. No. 08/017,683, filed on Feb. 12, 1993 and now U.S. Pat. No.5,305,757, which in turn is a Divisional of Ser. No. 717,084, filed onJun. 18, 1991 and now U.S. Pat. No. 5,228,446, which acontinuation-in-part of application U.S. Ser. No. 569,828, filed Aug.20, 1990 and now U.S. Pat. No. 5,088,499, which in turn is acontinuation-in-part of application U.S. Ser. No. 455,707, filed Dec.22, 1989 and now abandoned, the disclosures of each of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of ultrasonic imaging and, morespecifically, to gas filled liposomes prepared using vacuum drying gasinstillation methods, and to gas filled liposomes substantially devoidof liquid in the interior thereof. The invention also relates to methodsof and apparatus for preparing such liposomes and to methods foremploying such liposomes in ultrasonic imaging applications.

2. Background of the invention

There are a variety of imaging techniques which have been used to detectand diagnose disease in animals and humans. One of the first techniquesused for diagnostic imaging was X-rays. The images obtained through thistechnique reflect the electron density of the object being imaged.Contrast agents such as barium or iodine have been used over the yearsto attenuate or block X-rays such that the contrast between variousstructures is increased. For example, barium is used forgastro-intestinal studies to define the bowel lumen and visualize themucosal surfaces of the bowel. Iodinated contrast media is usedintravascularly to visualize the arteries in an X-ray process calledangiography. X-rays, however, are known to be somewhat dangerous, sincethe radiation employed in X-rays is ionizing, and the variousdeleterious effects of ionizing radiation are cumulative.

Magnetic resonance imaging (MRI) is another important imaging technique,however this technique has various drawbacks such as expense and thefact that it cannot be conducted as a portable examination. In addition,MRI is not available at many medical centers.

Radionuclides, employed in nuclear medicine, provide a further imagingtechnique. In employing this technique, radionuclides such as technetiumlabelled compounds are injected into the patient, and images areobtained from gamma cameras. Nuclear medicine techniques, however,suffer from poor spatial resolution and expose the animal or patient tothe deleterious effects of radiation. Furthermore, there is a problemwith the handling and disposal of radionuclides.

Ultrasound, a still further diagnostic imaging technique, is unlikenuclear medicine and X-rays in that it does not expose the patient tothe harmful effects of ionizing radiation. Moreover, unlike magneticresonance imaging, ultrasound is relatively inexpensive and can beconducted as a portable examination. In using the ultrasound technique,sound is transmitted into a patient or animal via a transducer. When thesound waves propagate through the body, they encounter interfaces fromtissues and fluids. Depending on the acoustic properties of the tissuesand fluids in the body, the ultrasound sound waves are partially orwholly reflected or absorbed. When sound waves are reflected by aninterface they are detected by the receiver in the transducer andprocessed to form an image. The acoustic properties of the tissues andfluids within the body determine the contrast which appears in theresultant image.

Advances have been made in recent years in ultrasound technology.However, despite these various technological improvements, ultrasound isstill an imperfect tool in a number of respects, particularly withregard to the imaging and detection of disease in the liver and spleen,kidneys, heart and vasculature, including measuring blood flow. Theability to detect and measure these things depends on the difference inacoustic properties between tissues or fluids and the surroundingtissues or fluids. As a result, contrast agents have been sought whichwill increase the acoustic difference between tissues or fluids and thesurrounding tissues or fluids in order to improve ultrasonic imaging anddisease detection.

The principles underlying image formation in ultrasound have directedresearchers to the pursuit of gaseous contrast agents. Changes inacoustic properties or acoustic impedance are most pronounced atinterfaces of different substances with greatly differing density oracoustic impedance, particularly at the interface between solids,liquids and gases. When ultrasound sound waves encounter suchinterfaces, the changes in acoustic impedance result in a more intensereflection of sound waves and a more intense signal in the ultrasoundimage. An additional factor affecting the efficiency or reflection ofsound is the elasticity of the reflecting interface. The greater theelasticity of this interface, the more efficient the reflection ofsound. Substances such as gas bubbles present highly elastic interfaces.Thus, as a result of the foregoing principles, researchers have focusedon the development of ultrasound contrast agents based on gas bubbles orgas containing bodies. However, despite the theoretical reasons why suchcontrast agents should be effective, overall the diagnostic results todate have been somewhat disappointing.

New and/or better contrast agents for ultrasound imaging are needed. Thepresent invention is directed to addressing these and/or other importantreeds.

SUMMARY OF THE INVENTION

The present invention provides contrast agents for ultrasonic imaging.

Specifically, in one embodiment, the present invention providesultrasound contrast agents comprising gas filled liposomes prepared byvacuum drying gas instillation methods, such liposomes sometimes beingreferred to herein as vacuum dried gas instilled liposomes.

In another embodiment, the invention is directed to contrast agentscomprising gas filled liposomes substantially devoid of liquid in theinterior thereof.

In a further embodiment, the subject invention provides methods forpreparing the liposomes of the subject invention, said methodscomprising: (i) placing liposomes under negative pressure; (ii)incubating the liposomes under the negative pressure for a timesufficient to remove substantially all liquid from the liposomes; and(iii) instilling selected gas into the liposomes until ambient pressuresare achieved. Methods employing the foregoing steps are referred toherein as the vacuum drying gas instillation methods.

In a still further embodiment, the invention provides apparatus forpreparing the liposomes of the invention using the vacuum drying gasinstillation methods, said apparatus comprising: (i) a vessel containingliposomes; (ii) means for applying negative pressure to the vessel todraw liquid from the liposomes contained therein; (iii) a conduitconnecting the negative pressurizing means to the vessel, the conduitdirecting the flow of said liquid; and (iv) means for introducing a gasinto the liposomes in the vessel.

In additional embodiments, the invention contemplates methods forproviding an image of an internal region of a patient, and/or fordiagnosing the presence of diseased tissue in a patient, comprising: (i)administering to the patient the liposomes of the present invention; and(ii) scanning the patient using ultrasonic imaging to obtain visibleimages of the region of the patient, and/or of any diseased tissue inthe patient.

Finally, the present invention provides diagnostic kits for ultrasonicimaging which include the contrast agents of the invention.

Surprisingly, the gas filled liposomes prepared by the vacuum drying gasinstillation method, and the gas filled liposomes substantially devoidof liquid in the interior thereof which may be prepared in accordancewith the vacuum drying gas instillation method, possess a number ofunexpected, but highly beneficial, characteristics. The liposomes of theinvention exhibit intense echogenicity on ultrasound, are highly stableto pressure and/or possess a long storage life, either when stored dryor suspended in a liquid medium. Also surprising is the ability of theliposomes during the vacuum drying gas instillation process to fill withgas and resume their original circular shape, rather than irreversiblycollapse into a cup-like shape.

Indeed, despite the theoretical reasons why the prior art ultrasoundcontrast agents based on gas bubbles or gas containing bodies should beeffective, the diagnostic results had remained largely disappointing. Anumber of the gaseous ultrasound contrast agents developed by priorresearchers involved unstabilized bubbles, and it has been found thatthe instability of these contrast agents severely diminishes thediagnostic usefulness of such agents. Other gaseous ultrasound contrastagents developed by prior researchers have involved gas bubblesstabilized in constructs which also contain a substantial amount ofliquid, and it has been found that the presence of a substantial amountof liquid in the construct leads to less satisfactory diagnosticresults. Indeed, the presence of liquid in the construct has been foundto disadvantageously alter the resonant characteristics of the gas inthe construct, and has been found to hasten the diffusion of furtherliquid into (and concomitantly gas out of) the construct. The presentinvention provides new and/or better contrast agents for ultrasoundimaging in an effort to address these and/or other important needs.

These and other features of the invention and the advantages thereofwill be set forth in greater detail in the figures and the descriptionbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an apparatus according to the present invention forpreparing the vacuum dried gas instilled liposomes and the gas filledliposomes substantially devoid of liquid in the interior thereofprepared by the vacuum drying gas instillation method.

FIG. 2 is a graphical representation of the dB reflectivity of gasfilled liposomes substantially devoid of liquid in the interior thereofprepared by the vacuum drying gas instillation method. The data wasobtained by scanning with a 7.5 megahertz transducer using an AcousticImaging™ Model 5200 scanner (Acoustic Imaging, Phoenix, Ariz.), and wasgenerated by using the system test software to measure reflectivity. Thesystem was standardized prior to each experiment with a phantom of knownacoustic impedance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ultrasound contrast agentscomprising gas filled liposomes prepared by vacuum drying gasinstillation methods, such liposomes sometimes being referred to hereinas vacuum dried gas instilled liposomes. The present invention isfurther directed to contrast agents comprising gas filled liposomessubstantially devoid of liquid in the interior thereof.

The vacuum drying gas instillation method which may be employed toprepare both the gas filled liposomes prepared by the vacuum drying gasinstillation method, and the gas filled liposomes substantially devoidof liquid in the interior thereof, contemplates the following process.First, in accordance with the process, the liposomes are placed undernegative pressure (that is, reduced pressure or vacuum conditions).Next, the liposomes are incubated under that negative pressure for atime sufficient to remove substantially all liquid from the liposomes,thereby resulting in substantially dried liposomes. By removal ofsubstantially all liquid, and by substantially dried liposomes, as thosephrases are used herein, it is meant that the liposomes are at leastabout 90% devoid of liquid, preferably at least about 95% devoid ofliquid, most preferably about 100% devoid of liquid. Finally, theliposomes arc instilled with selected gas by applying the gas to theliposomes until ambient pressures are achieved, thus resulting in thesubject vacuum dried gas instilled liposomes of the present invention,and the gas filled liposomes of the invention substantially devoid ofliquid in the interior thereof. By substantially devoid of liquid in theinterior thereof, as used herein, it is meant liposomes having aninterior that is at least about 90% devoid of liquid, preferably atleast about 95% devoid of liquid, most preferably about 100% devoid ofliquid.

Unexpectedly, the liposomes prepared in accordance with the vacuum driedgas instillation method, and the gas filled liposomes, substantiallydevoid of liquid in the interior thereof, possess a number of surprisingyet highly beneficial characteristics. The liposomes of the inventionexhibit intense echogenicity on ultrasound, are highly stable topressure, and/or generally possess a long storage life, either whenstored dry or suspended in a liquid medium. The ecogenicity of theliposomes is of obvious importance to the diagnostic applications of theinvention, and is illustrated in FIG. 2. Preferably, the liposomes ofthe invention possess a reflectivity of greater than 2 dB, preferablybetween about 4 dB and about 20 dB. Within these ranges, the highestreflectivity for the liposomes of the invention is exhibited by thelarger liposomes, by higher concentrations of liposomes, and/or wherehigher ultrasound frequencies are employed. The stability of theliposomes is also of great practical importance. The subject liposomestend to have greater stability during storage than other gas filledliposomes produced via known procedures such as pressurization or othertechniques. At 72 hours after formation, for example, conventionallyprepared liposomes often are essentially devoid of gas, the gas havingdiffused out of the liposomes and/or the liposomes having rupturedand/or fused, resulting in a concomitant loss in reflectivity. Incomparison, gas filled liposomes of the present invention generally havea shelf life stability of greater than about three weeks, preferably ashelf life stability of greater than about four weeks, more preferably ashelf life stability of greater than about five weeks, even morepreferably a shelf life stability of greater than about three months,and often a shelf life stability that is even much longer, such as oversix months, twelve months, or even two years.

Also unexpected is the ability of the liposomes during the vacuum dryinggas instillation process to fill with gas and resume their originalcircular shape, rather than collapse into a cup-shaped structure, as theprior art would cause one to expect. See, e.g., Crowe et al., Archivesof Biochemistry and Biophysics, Vol. 242, pp. 240-247 (1985); Crowe etal., Archives of Biochemistry and Biophysics, Vol. 220, pp. 477-484(1983); Fukuda et al., J. Am. Chem. Soc., Vol. 108, pp. 2321-2327(1986); Regen et al., J. Am. Chem. Soc., Vol. 102, pp. 6638-6640 (1980).

The liposomes subjected to the vacuum drying gas instillation method ofthe invention may be prepared using any one of a variety of conventionalliposome preparatory techniques which will be apparent to those skilledin the art. These techniques include freeze-thaw, as well as techniquessuch as sonication, chelate dialysis, homogenization, solvent infusion,microemulsification, spontaneous formation, solvent vaporization, Frenchpressure cell technique, controlled detergent dialysis, and others. Thesize of the liposomes can be adjusted, if desired, prior to vacuumdrying and gas instillation, by a variety of procedures includingextrusion, filtration, sonication, homogenization, employing a laminarstream of a core of liquid introduced into an immiscible sheath ofliquid, and similar methods, in order to modulate resultant liposomalbiodistribution and clearance. Extrusion under pressure through pores ofdefined size is, however, the preferred means of adjusting the size ofthe liposomes. The foregoing techniques, as well as others, arediscussed, for example, in U.S. Pat. No. 4,728,578; U.K. PatentApplication GB 2193095 A; U.S. Pat. No. 4,728,575; U.S. Pat. No.4,737,323; International Application PCT/US85/01161; Mayer et al.,Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986); Hope etal., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985); U.S.Pat. No. 4,533,254; Mayhew et al., Methods in Enzymology, Vol. 149, pp.64-77 (1987); Mayhew et al., Biochimica et Biophysica Acta, Vol 755, pp.169-74 (1984); Cheng et al, Investigative Radiology, Vol. 22, pp. 47-55(1987); PCT/US89/05040; U.S. Pat. No. 4,162,282; U.S. Pat. No.4,310,505; U.S. Pat. No. 4,921,706; and Liposomes Technology,Gregoriadis, G., ed., Vol. I, pp. 29-37, 51-67 and 79-108 (CRC PressInc, Boca Raton, Fla., 1984). The disclosures of each of the foregoingpatents, publications and patent applications are incorporated byreference herein, in their entirety. Although any of a number of varyingtechniques can be employed, preferably the liposomes are prepared viamicroemulsification techniques. The liposomes produced by the variousconventional procedures can then be employed in the vacuum drying gasinstillation method of the present invention, to produce the liposomesof the present invention.

The materials which may be utilized in preparing liposomes to beemployed in the vacuum drying gas instillation method of the presentinvention include any of the materials or combinations thereof known tothose skilled in the art as suitable for liposome construction. Thelipids used may be of either natural or synthetic origin. Such materialsinclude, but are not limited to, lipids such as fatty acids, lysolipids,dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidic acid,sphingomyelin, cholesterol, cholesterol hemisuccinate, tocopherolhemisuccinate, phosphatidylethanolamine, phosphatidyl-inositol,lysolipids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether and ester-linked fatty acids, polymerizedlipids, diacetyl phosphate, stearylamine, distearoylphosphatidylcholine,phosphatidylserine, sphingomyelin, cardiolipin, phospholipids with shortchain fatty acids of 6-8 carbons in length, synthetic phospholipids withasymmetric acyl chains (e.g., with one acyl chain of 6 carbons andanother acyl chain of 12 carbons),6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside,digalactosyldiglyceride,6-(5cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside,dibehenoyl-phosphatidylcholine, dimyristoylphosphatidylcholine,dilauroylphosphatidylcholine, and dioleoyl-phosphatidylcholine, and/orcombinations thereof. Other useful lipids or combinations thereofapparent to those skilled in the art which are in keeping with thespirit of the present invention are also encompassed by the presentinvention. For example, carbohydrates bearing lipids may be employed forin vivo targeting as described in U.S. Pat. No. 4,310,505. Of particularinterest for use in the present invention are lipids which are in thegel state (as compared with the liquid crystalline state) at thetemperature at which the vacuum drying gas instillation is performed.The phase transition temperatures of various lipids will be readilyapparent to those skilled in the art and are described, for example, inLiposome Technology, Gregoriadis, G., ed., Vol. I, pp. 1-18 (CRC Press,Inc. Boca Raton, Fla. 1984), the disclosures of which are incorporatedherein by reference in their entirety. In addition, it has been foundthat the incorporation of at least a small amount of negatively chargedlipid into any liposome membrane, although not required, is beneficialto providing highly stable liposomes. By at least a small amount, it ismeant about 1 mole percent of the total lipid. Suitable negativelycharged lipids will be readily apparent to those skilled in the art, andinclude, for example phosphatidylserine and fatty acids. Most preferredfor reasons of the combined ultimate ecogenicity and stability followingthe vacuum drying gas instillation process are liposomes prepared fromdipalmitoyl-phosphatidylcholine.

By way of general guidance, dipalmitoyl-phosphatidylcholine liposomesmay be prepared by suspending dipalmitoylphosphatidylcholine lipids inphosphate buffered saline or water, and heating the lipids to about 50°C., a temperature which is slightly above the 45° C. temperaturerequired for transition of the dipalmitoyl-phosphatidylcholine lipidsfrom a gel state to a liquid crystalline state, to form liposomes. Toprepare multilamellar vesicles of a rather heterogeneous sizedistribution of around 2 microns, the liposomes may then be mixed gentlyby hand while keeping the liposome solution at a temperature of about50° C. The temperature is then lowered to room temperature, and theliposomes remain intact. Extrusion of dipalmitoylphosphatidylcholineliposomes through polycarbonate filters of defined size may, if desired,be employed to make liposomes of a more homogeneous size distribution. Adevice useful for this technique is an extruder device (ExtruderDevice™, Lipex Biomembranes, Vancouver, Canada) equipped with a thermalbarrel so that extrusion may be conveniently accomplished above the gelstate-liquid crystalline transition temperature for lipids.

Alternatively, and again by way of general guidance, conventionalfreeze-thaw procedures may be used to produce either oligolamellar orunilamellar dipalmitoyl-phosphatidylcholine liposomes. After thefreeze-thaw procedures, extrusion procedures as described above may thenbe performed on the liposomes.

The liposomes thus prepared may then be subjected to the vacuum dryinggas instillation process of the present invention, to produce the vacuumdried gas instilled liposomes, and the gas filled liposomessubstantially devoid of liquid in the interior thereof, of theinvention. In accordance with the process of the invention, theliposomes are placed into a vessel suitable for subjecting to theliposomes to negative pressure (that is, reduced pressure or vacuumconditions). Negative pressure is then applied for a time sufficient toremove substantially all liquid from the liposomes, thereby resulting insubstantially dried liposomes. As those skilled in the art wouldrecognize, once armed with the present disclosure, various negativepressures can be employed, the important parameter being thatsubstantially all of the liquid has been removed from the liposomes.Generally, a negative pressure of at least about 700 mm Hg andpreferably in the range of between about 700 mm Hg and about 760 mm Hg(gauge pressure), applied for about 24 to about 72 hours, is sufficientto remove substantially all of the liquid from the liposomes. Othersuitable pressures and time periods will be apparent to those skilled inthe art, in view of the disclosures herein.

Finally, a selected gas is applied to the liposomes to instill theliposomes with gas until ambient pressures are achieved, therebyresulting in the vacuum dried gas instilled liposomes of the invention,and in the gas filled liposomes substantially devoid of liquid in theinterior thereof. Preferably, gas instillation occurs slowly, that is,over a time period of at least about 4 hours, most preferably over atime period of between about 4 and about 8 hours. Various biocompatiblegases may be employed. Such gases include air, nitrogen, carbon dioxide,oxygen, argon, xenon, neon, helium, or any and all combinations thereof.Other suitable gases will be apparent to those skilled in the art, thegas chosen being only limited by the proposed application of theliposomes.

The above described method for production of liposomes is referred tohereinafter as the vacuum drying gas instillation process.

If desired, the liposomes may be cooled, prior to subjecting theliposomes to negative pressure, and such cooling is preferred.Preferably, the liposomes are cooled to below 0° C., more preferably tobetween about -10° C. and about -20° C., and most preferably to -10° C.,prior to subjecting the liposomes to negative pressure. Upon reachingthe desired negative pressure, the liposomes temperature is thenpreferably increased to above 0° C., more preferably to between about10° C. and about 20° C., and most preferably to 10° C., untilsubstantially all of the liquid has been removed from the liposomes andthe negative pressure is discontinued, at which time the temperature isthen permitted to return to room temperature.

If the liposomes are cooled to a temperature below 0° C., it ispreferable that the vacuum drying gas instillation process be carriedout with liposomes either initially prepared in the presence ofcryoprotectants, or liposomes to which cryoprotectants have been addedprior to carrying out the vacuum drying gas instillation process of theinvention. Such cryoprotectants, while not mandatorily added, assist inmaintaining the integrity of liposome membranes at low temperatures, andalso add to the ultimate stability of the membranes. Preferredcryoprotectants are trehalose, glycerol, polyethyleneglycol (especiallypolyethyleneglycol of molecular weight 400), raffinose, sucrose andsorbitol, with trehalose being particularly preferred.

It has also been surprisingly discovered that the liposomes of theinvention are highly stable to changes in pressure. Because of thischaracteristic, extrusion of the liposomes through filters of definedpore size following vacuum drying and gas instillation can be carriedout, if desired, to create liposomes of relatively homogeneous anddefined pore size.

For storage prior to use, the liposomes of the present invention may besuspended in an aqueous solution, such as a saline solution (forexample, a phosphate buffered saline solution), or simply water, andstored preferably at a temperature of between about 2° C. and about 10°C., preferably at about 4° C. Preferably, the water is sterile. Mostpreferably, the liposomes are stored in a hypertonic saline solution(e.g., about 0.3 to about 0.5% NaCl), although, if desired, the salinesolution may be isotonic. The solution also may be buffered, if desired,to provide a pH range of pH 6.8 to pH 7.4. Suitable buffers include, butare not limited to, acetate, citrate, phosphate and bicarbonate.Dextrose may also be included in the suspending media. Preferably, theaqueous solution is degassed (that is, degassed under vacuum pressure)prior to suspending the liposomes therein. Bacteriostatic agents mayalso be included with the liposomes to prevent bacterial degradation onstorage. Suitable bacteriostatic agents include but are not limited tobenzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, butylparaben, cetylpyridinium chloride, chlorobutanol,chlorocresol, methylparaben, phenol, potassium benzoate, potassiumsorbate, sodium benzoate and sorbic acid. One or more antioxidants mayfurther be included with the gas filled liposomes to prevent oxidationof the lipid. Suitable antioxidants include tocopherol, ascorbic acidand ascorbyl palmitate. Liposomes prepared in the various foregoingmanners may be stored for at least several weeks or months. Liposomes ofthe present invention may alternatively, if desired, be stored in theirdried, unsuspended form, and such liposomes also have a shelf life ofgreater than several weeks or months. Specifically, the liposomes of thepresent invention, stored either way, generally have a shelf lifestability of greater than about three weeks, preferably a shelf lifestability of greater than about four weeks, more preferably a shelf lifestability of greater than about five weeks, even more preferably a shelflife stability of greater than about three months, and often a shelflife stability that is even much longer, such as over six months, twelvemonths or even two years.

As another aspect of the invention, useful apparatus for preparing thevacuum dried gas instilled liposomes, and the gas filled liposomessubstantially devoid of liquid in the interior thereof, of the inventionis also presented. Specifically, there is shown in FIG. 1 a preferredapparatus for vacuum drying liposomes and instilling a gas into thedried liposomes. The apparatus is comprised of a vessel 8 for containingliposomes 19. If desired, the apparatus may include an ice bath 5containing dry ice 17 surrounding the vessel 8. The ice bath 5 and dryice 17 allow the liposomes to be cooled to below 10° C. A vacuum pump 1is connected to the vessel 8 via a conduit 15 for applying a sustainednegative pressure to the vessel. In the preferred embodiment, the pump 1is capable of applying a negative pressure of at least about 700 mm Hg,and preferably a negative pressure in the range of about 700 mm Hg toabout 760 mm Hg (gauge pressure). A manometer 6 is connected to theconduit 15 to allow monitoring of the negative pressure applied to thevessel 8.

In order to prevent liquid removed from the liposomes from entering thepump 1, a series of traps are connected to the conduit 15 to assist incollecting the liquid (and liquid vapor, all collectively referred toherein as liquid) drawn from the liposomes. In a preferred embodiment,two traps are utilized. The first trap is preferably comprised of aflask 7 disposed in an ice bath 4 with dry ice 17. The second trap ispreferably comprised of a column 3 around which tubing 16 is helicallyarranged. The column 3 is connected to the conduit 15 at its top end andto one end of the tubing 16 at its bottom end. The other end of thetubing 16 is connected to the conduit 15. As shown in FIG. 1, an icebath 2 with dry ice 17 surrounds the column 3 and tubing 16. If desired,dry ice 17 can be replaced with liquid nitrogen, liquid air or othercryogenic material. The ice baths 2 and 4 assist in collecting anyliquid and condensing any liquid vapor drawn from the liposomes forcollection in the traps. In preferred embodiments of the presentinvention the ice traps 2 and 4 are each maintained at a temperature ofleast about -70° C.

A stopcock 14 is disposed in the conduit 15 upstream of the vessel 8 toallow a selected gas to be introduced into the vessel 8 and into theliposomes 19 from gas bottle 18.

Apparatus of the present invention are utilized by placing the liposomes19 into vessel 8. In a preferable embodiment, ice bath 5 with dry ice 17is used to lower the temperature of the liposomes to below 0° C., morepreferably to between about -10C and about -20° C., and most preferablyto -10° C. With stopcocks 14 and 9 closed, vacuum pump 1 is turned on.Stopcocks 10, 11, 12 and 13 are then carefully opened to create a vacuumin vessel 8 by means of vacuum pump 1. The pressure is gauged by meansof manometer 6 until negative pressure of at least about 700 mm Hg andpreferably in the range of between about 700 mn Hg and about 760 mm Hg(gauge pressure) is achieved. In preferred embodiments of the presentinvention vessel 7, cooled by ice bath 4 with dry ice 17, and column 3and coil 16, cooled by ice bath 2 with dry ice 17, together orindividually condense liquid vapor and trap liquid drawn from theliposomes so as to prevent such liquids and liquid vapor from enteringthe vacuum pump 1. In preferred embodiments of the present invention,the temperature of ice traps 2 and 4 are each maintained at atemperature of at least about -70° C. The desired negative pressure isgenerally maintained for at least 24 hours as liquid and liquid vapor isremoved from the liposomes 19 in vessel 8 and frozen in vessels 3 and 7.Pressure within the system is monitored using manometer 6 and isgenerally maintained for about 24 to about 72 hours, at which timesubstantially all of the liquid has been removed from the liposomes. Atthis point, stopcock 10 is slowly closed and vacuum pump 1 is turnedoff. Stopcock 14 is then opened gradually and gas is slowly introducedinto the system from gas bottle 18 through stopcock 14 via conduit 15 toinstill gas into the liposomes 19 in vessel 8. Preferably the gasinstillation occurs slowly over a time period of at least about 4 hours,most preferably over a time period of between about 4 and about 8 hours,until the system reaches ambient pressure.

The vacuum dried gas instilled liposomes and the gas filled liposomessubstantially devoid of liquid in the interior thereof of the presentinvention have superior characteristics for ultrasound contrast imaging.Specifically, the present invention is useful in imaging a patientgenerally, and/or in diagnosing the presence of diseased tissue in apatient. The patient may be any type of mammal, but is most preferablyhuman. Thus, in further embodiments of the present invention, a methodof providing an image of an internal bodily region of a patient isprovided. This method comprises administering the liposomes of theinvention to the patient and scanning the patient using ultrasonicimaging to obtain visible images of the region. A method is alsoprovided for diagnosing the presence of diseased tissue in a patient,said method comprising administering to a patient liposomes of thepresent invention, and then scanning the patient using ultrasonicimaging to obtain visible images of any diseased tissue in the patient.By region of a patient, it is meant the whole patient, or a particulararea or portion of the patient. For example, by using the method of theinvention, a patient's heart, and a patient's vasculature (that is,venous or arterial systems), may be visualized and/or diseased tissuemay be diagnosed. In visualizing a patient's vasculature, blood flow maybe measured, as will be well understood by those skilled in the art inview of the present disclosure. The invention is also particularlyuseful in visualizing and/or diagnosing disease in a patient's rightheart, a region not easily imaged heretofore by ultrasound. Liver,spleen and kidney regions of a patient may also be readily visualizedand/or disease detected therein using the present methods.

Liposomes of the present invention may be of varying sizes, butpreferably are of a size range wherein they have a mean outside diameterbetween about 30 nanometers and about 10 microns, with the preferablemean outside diameter being about 2 microns. As is known to thoseskilled in the art, liposome size influences biodistribution and,therefore, different size liposomes may be selected for variouspurposes. For intravascular use, for example, liposome size is generallyno larger than about 5 microns, and generally no smaller than about 30nanometers, in mean outside diameter. To provide ultrasound enhancementof organs such as the liver and to allow differentiation of tumor fromnormal tissue, smaller liposomes, between about 30 nanometers and about100 nanometers in mean outside diameter, are useful.

Any of the various types of ultrasound imaging devices can be employedin the practice of the invention, the particular type or model of thedevice not being critical to the method of the invention. Generally, forthe diagnostic uses of the present invention, ultrasound frequenciesbetween about 3.0 to about 7.5 megahertz are employed.

As one skilled in the art would recognize, administration of contrastimaging agents of the present invention may be carried out in variousfashions, such as intravascularly, intralymphatically, parenterally,subcutaneously, intramuscularly, intraperitoneally, interstitially,hyperbarically, orally, or intratumorly using a variety of dosage forms.One preferred route of administration is intravascularly. Forintravascular use the contrast agent is generally injectedintravenously, but may be injected intraarterially as well. The usefuldosage to be administered and the mode of administration will varydepending upon the age, weight, and mammal to be diagnosed, and theparticular diagnostic application intended. Typically dosage isinitiated at lower levels and increased until the desired contrastenhancement is achieved. Generally, the contrast agents of the inventionare administered in the form of an aqueous suspension such as in wateror a saline solution (e.g., phosphate buffered saline). Preferably, thewater is sterile. Also preferably the saline solution is a hypertonicsaline solution (e.g., about 0.3 to about 0.5% NaCl), although, ifdesired, the saline solution may be isotonic. The solution also may bebuffered, if desired, to provide a pH range of pH 6.8 to pH 7.4. Inaddition, dextrose may be preferably included in the media. Preferably,the aqueous solution is degassed (that is, degassed under vacuumpressure) prior to suspending the liposomes therein.

Kits useful for ultrasonic imaging in accordance with the presentinvention comprise gas filled liposomes prepared by a vacuum drying gasinstillation methods, and gas filled liposomes substantially devoid ofliquid in the interior thereof, in addition to conventional ultrasonicimaging kit components. Such conventional ultrasonic imaging kitcomponents are well known, and include, for example, filters to removebacterial contaminants or to break up liposomal aggregates prior toadministration.

The liposomes of the present invention are believed to differ from theliposomes of the prior art in a number of respects, both in physical andin functional characteristics. For example, the liposomes of theinvention are substantially devoid of liquid in the interior thereof. Bydefinition, liposomes in the prior art have been characterized by thepresence of an aqueous medium. See, e.g., Dorland's Illustrated MedicalDictionary, p. 946, 27th ed. (W. B. Saunders Company, Philadelphia1988). Moreover, the present liposomes surprisingly exhibit intenseecogenicity on ultrasound, and possess a long storage life,characteristics of great benefit to the use of the liposomes asultrasound contrast agents.

There are various other applications for liposomes of the invention,beyond those described in detail herein. Such additional uses, forexample, include such applications as hyperthermia potentiators forultrasound and as drug delivery vehicles. Such additional uses and otherrelated subject matter are described and claimed in applicant's patentapplications filed concurrently herewith entitled "Novel Liposomal DrugDelivery Systems" and "Method For Providing Localized Therapeutic Heatto Biological Tissues and Fluids Using Gas Filled Liposomes", thedisclosures of each of which are incorporated herein by reference intheir entirety.

The present invention is further described in the following examples.Examples 1-8 are actual examples that describe the preparation andtesting of the vacuum dried gas instilled liposomes, the gas filledliposomes being substantially devoid of any liquid in the interiorthereof. Examples 9-11 are prophetic examples that describe the use ofthe liposomes of the invention. The following examples should not beconstrued as limiting the scope of the appended claims.

EXAMPLES Example 1

Dipalmitoylphosphatidylcholine (1 gram) was suspended in 10 ml phosphatebuffered saline, the suspension was heated to about 50° C., and thenswirled by hand in a round bottom flask for about 30 minutes. The heatsource was removed, and the suspension was swirled for two additionalhours, while allowing the suspension to cool to room temperature, toform liposomes.

The liposomes thus prepared were placed in a vessel in an apparatussimilar to that shown in FIG. 1, cooled to about -10° C, and thensubjected to high negative vacuum pressure. The temperature of theliposomes was then raised to about 10° C. High negative vacuum pressurewas maintained for about 48 hours. After about 48 hours, nitrogen gaswas gradually instilled into the chamber over a period of about 4 hours,after which time the pressure returned to ambient pressure. Theresulting vacuum dried gas instilled liposomes, the gas filled liposomesbeing substantially devoid of any liquid in the interior thereof, werethen suspended in 10 cc of phosphate buffered saline and stored at about4° C. for about three months.

Example 2

To test the liposomes of Example 1 ultrasonographically, a 250 mg sampleof these liposomes was suspended in 300 cc of degassed phosphatebuffered saline (that is, degassed under vacuum pressure). The liposomeswere then scanned in vitro at varying time intervals with a 7.5 mHztransducer using an Acoustic Imaging Model 5200 scanner (AcousticImaging, Phoenix, Ariz) and employing the system test software tomeasure dB reflectivity. The system was standardized prior to testingthe liposomes with a phantom of known acoustic impedance. A graphshowing dB reflectivity is provided in FIG. 2.

Example 3

Dipalmitoylphosphatidylcholine (1 gram) and the cryoprotectant trehalose(1 gram) were suspended in 10 ml phosphate buffered saline, thesuspension was heated to about 50° C., and then swirled by hand in around bottom flask for about 30 minutes. The heat source was removed,and the suspension was swirled for about two additional hours, whileallowing the suspension to cool to room temperature, to form liposomes.

The liposomes thus prepared were then vacuum dried and gas instilled,substantially following the procedures shown in Example 1, resulting invacuum dried gas instilled liposomes, the gas filled liposomes beingsubstantially devoid of any liquid in the interior thereof. Theliposomes were then suspended in 10 cc of phosphate buffered saline, andthen stored at about 4° C. for several weeks.

Example 4

To test the liposomes of Example 3 ultrasonographically, the proceduresof Example 2 were substantially followed. The dB reflectivity of theliposomes were similar to the dB reflectivity reported in Example 2.

Example 5

Dipalmitoylphosphatidylcholine (1 gram) was suspended in 10 ml phosphatebuffered saline, the suspension was heated to about 50° C., and thenswirled by hand in a round bottom flask for about 30 minutes. Thesuspension was then subjected to 5 cycles of extrusion through anextruder device jacketed with a thermal barrel (Extruder Device™, LipexBiomembranes, Vancouver, Canada), both with and without conventionalfreeze-thaw treatment prior to extrusion, while maintaining thetemperature at about 50° C. The heat source was removed, and thesuspension was swirled for about two additional hours, while allowingthe suspension to cool to room temperature, to form liposomes.

The liposomes thus prepared were then vacuum dried and gas instilled,substantially following the procedures shown in Example 1, resulting invacuum dried gas instilled liposomes, the gas filled liposomes beingsubstantially devoid of any liquid in the interior thereof. Theliposomes were then suspended in 10 cc of phosphate buffered saline, andthen stored at about 4° C. for several weeks.

Example 6

To test the liposomes of Example 5 ultrasonographically, the proceduresof Example 2 were substantially followed. The dB reflectivity of theliposomes were similar to the dB reflectivity reported in Example 2.

Example 7

In order to test the stability of the liposomes of the invention, theliposomes suspension of Example 1 was passed through 2 micronpolycarbonate filters in an extruder device (Extruder Device™, LipexBiomembranes, Vancouver, Canada) five times at a pressure of about 600psi. After extrusion treatment, the liposomes were studiedultrasonographically, as described in Example 2. surprisingly, evenafter extrusion under high pressure, the liposomes of the inventionsubstantially retained their echogenicity.

Example 8

The liposomes of Example 1 were scanned by ultrasound using transducerfrequencies varying from 3 to 7.5 mHz. The results indicated that at ahigher frequency of ultrasound, the echogenicity decays more rapidly,reflecting a relatively high resonant frequency and higher energyassociated with the higher frequencies.

The following examples, Examples 9, 10, and 11, are prophetic examples.

Example 9

A patient with suspected myocardial ischemia is administered anintravenous dose of 500 mg of vacuum dried gas instilled liposomesencapsulating nitrogen gas, the gas filled liposomes being substantiallydevoid of liquid in the interior thereof, with a mean diameter of 2microns, and the left ventricular myocardium is studiedultrasonographically.

Example 10

A patient with suspected myocardial ischemia is administered anintravenous dose of 500 mg of vacuum dried gas instilled liposomesencapsulating nitrogen gas, the gas filled liposomes being substantiallydevoid of liquid in the interior thereof, with a mean diameter of 2microns, and the left ventricular myocardium is studiedultrasonographically.

Example 11

A patient with suspected hepatic metastases is administered anintravenous dose of 500 mg of vacuum dried gas instilled liposomesencapsulating nitrogen gas, the gas filled liposomes being substantiallydevoid of liquid in the interior thereof, and the liver is examinedultrasonographically.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A gas filled liposome prepared by a vacuum dryinggas instillation method.
 2. A gas filled liposome of claim 1 whereinsaid liposome is comprised of lipid materials selected from the groupconsisting of fatty acids, lysolipids, dipalmitoylphosphatidylcholine,phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol,cholesterol hemisuccinate, tocopherol hemisuccinate,phosphatidylethanolamine, phosphatidylinositol, lysolipids,sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether and ester-linked fatty acids, andpolymerized lipids.
 3. A gas filled liposome of claim 2 wherein saidliposome is comprised of dipalmitoylphosphatidylcholine.
 4. A gas filledliposome of claim 2 wherein said liposome comprises polymerized lipids.5. A gas filled liposome of claim 2 further comprisingpolyethyleneglycol.
 6. A gas filled liposome of claim 2 wherein saidliposome is filled with a gas selected from the group consisting of air,nitrogen, carbon dioxide, oxygen, argon, xenon, helium, and neon.
 7. Agas filled liposome of claim 6 wherein said liposome is filled withnitrogen gas.
 8. A gas filled liposome of claim 1 wherein said liposomeis stored suspended in an aqueous medium.
 9. A gas filled liposome ofclaim 1 wherein said liposome is stored dry.
 10. A gas filled liposomeof claim 1 wherein said liposome has a stability of greater than aboutthree weeks.
 11. A gas filled liposome of claim 1 wherein said liposomehas a reflectivity of greater than about 2 dB.
 12. A gas filled liposomeof claim 11 wherein said liposome has a reflectivity of between about 2dB and about 20 dB.
 13. A gas filled liposome of claim 1 wherein saidliposome comprises a material for in vivo targeting.
 14. A gas filledliposome of claim 13 wherein said targeting material comprises acarbohydrate.
 15. A gas filled liposome of claim 1 wherein said liposomeis stable to changes in pressure.
 16. A gas filled liposome of claim 1wherein said liposome is selected from the group consisting ofoligolamellar liposomes and unilamellar liposomes.
 17. A gas filledliposome of claim 16 wherein said liposome comprises oligolamellarliposomes.
 18. A gas filled liposome of claim 16 wherein said liposomecomprises unilamellar liposomes.
 19. A gas filled liposome of claim 18which comprises a phospholipid.
 20. A gas filled liposome of claim 16which comprises a phospholipid.
 21. A gas filled liposome substantiallydevoid of liquid in the interior thereof.
 22. A gas filled liposome ofclaim 21 wherein said liposome is comprised of lipid materials selectedfrom the group consisting of fatty acids, lysolipids,dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidic acid,sphingomyelin, cholesterol, cholesterol hemisuccinate, tocopherolhemisuccinate, phosphatidylethanolamine, phosphatidylinositol,lysolipids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether and ester-linked fatty acids, andpolymerized lipids.
 23. A gas filled liposome of claim 22 wherein saidliposome is comprised of dipalmitoylphosphatidylcholine.
 24. A gasfilled liposome of claim 22 wherein said liposome comprises polymerizelipids.
 25. A gas filled liposome of claim 22 further comprisingpolyethyleneglycol.
 26. A gas filled liposome of claim 21 wherein saidliposome is filled with a gas selected from the group consisting of air,nitrogen, carbon dioxide, oxygen, argon, xenon, helium, and neon.
 27. Agas filled liposome of claim 26 wherein said liposome is filled withnitrogen gas.
 28. A gas filled liposome of claim 21 wherein saidliposome is stored suspended in an aqueous medium.
 29. A gas filledliposome of claim 21 wherein said liposome is stored dry.
 30. A gasfilled liposome of claim 21 wherein said liposome has a shelf lifestability of greater than about three weeks.
 31. A gas filled liposomeof claim 21 wherein said liposome has a reflectivity of greater thanabout 2 dB.
 32. A gas filled liposome of claim 31 wherein said liposomehas a reflectivity of between about 2 dB and about 20 dB.
 33. A gasfilled liposome of claim 21 wherein said liposome comprises a materialfor in vivo targeting.
 34. A gas filled liposome of claim 33 whereinsaid material comprises a carbohydrate.
 35. A gas filled liposome ofclaim 21 wherein said liposome is stable to changes in pressure.
 36. Agas filled liposome of claim 21 wherein said liposome is selected fromthe group consisting of oligolamellar liposomes and unilamellarliposomes.
 37. A gas filled liposome of claim 36 wherein said liposomecomprises oligolamellar liposomes.
 38. A gas filled liposome of claim 36wherein said liposome comprises unilamellar liposomes.
 39. A gas filledliposome of claim 38 which comprises a phospholipid.
 40. A gas filledliposome of claim 36 which comprises a phospholipid.
 41. A contrastagent adapted to be inject ed in to the body of a patient and comprisingstabilized gas bubbles which comprises gas encapsulated by one or morelipid materials, said stabilized gas bubbles being substantially devoidof liquid in the interior thereof.
 42. A contrast agent of claim 41wherein said stabilized gas bubbles comprise liposomes that are selectedfrom the group consisting of oligolamellar liposomes and unilamellarliposomes.
 43. A contrast agent of claim 42 wherein said stabilized gasbubbles comprise oligolamellar liposomes.
 44. A contrast agent of claim42 wherein said stabilized gas bubbles comprise unilamellar liposomes.45. A contrast agent of claim 44 wherein said lipid materials comprisephospholipids.
 46. A contrast agent of claim 42 wherein said lipidmaterials comprise phospholipids.
 47. A contrast agent of claim 41wherein said lipid materials are selected from the group consisting offatty acids, lysolipids, dipalmitoylphosphatidylcholine,phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol,cholesterol hemisuccinate, tocopherol hemisuccinate,phosphatidylethanolamine, phosphatidylinositol, lysolipids,sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether and ester-linked fatty acids, andpolymerized lipids.
 48. A contrast agent of claim 47 wherein said lipidmaterials comprise dipalmitoylphosphatidylcholine.
 49. A contrast agentof claim 47 wherein said lipid materials comprise polymerized lipids.50. A contrast agent of claim 41 further comprising polyethyleneglycol.51. A contrast agent of claim 41 which comprises a material for in vivotargeting.
 52. A contrast agent of claim 51 wherein said materialcomprises a carbohydrate.
 53. A contrast agent of claim 41 wherein saidgas is selected from the group consisting of air, nitrogen, carbondioxide, oxygen, argon, xenon, helium, and neon.
 54. A contrast agent ofclaim 41 wherein said stabilized gas bubbles are stored suspended in anaqueous medium.
 55. A contrast agent of claim 41 which is stored dry.56. A contrast agent of claim 41 wherein said stabilized gas bubbles arestable to changes in pressure.